This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; methods of making the polynucleotides and polypeptides, and their variants and derivatives; and uses of the polynucleotides, polypeptides, variants, and derivatives. In particular, in these and in other regards, the invention relates to novel human inhibitor-of-apoptosis polypeptides and the polynucleotides which encode these polypeptides.
Apoptosis, also known as programmed cell death, is a genetically controlled process which plays an important role in development and in cellular and tissue homeostasis (Hengartner, M., Exp. Gerontol. 32: 363-374, 1997; Hoeppner et al., Biochem. Biophys. Acta 1242:217-220, 1996; Ellis et al., Annual Rev. Cell Biol. 7:663-698, 1991). Apoptosis permits the elimination of cells which either have been overproduced, developed improperly or have undergone genetic damage and represents a major host defense mechanism for limiting the replication of infective viruses. In contrast to necrotic cell death, which is usually accompanied by swelling and disruption of cellular membranes and inflammation of adjacent tissue, apoptosis is marked by cell shrinkage, blebbing, chromatin condensation, DNA fragmentation and formation of apoptotic bodies (MacLellan and Schneider, Circ. Res. 81:137-144, 1997; Cohen, G., Biochem. J. 326 (Pt 1):1-16, 1997). Apoptotic cells are then phagocytosed by neighboring scavenger cells without eliciting an inflammatory response (Wu and Horvitz, Nature 392:501-504, 1998).
Deregulation of apoptosis has been implicated in the pathogenesis of a variety of diseases. Impaired apoptosis can play a role in cancer (Pan et al., Cancer Surv. 29:305-327, 1997; Thompson, C., Science 267:1456-1462, 1995) or chronic viral infection (Clem et al., Science 254:1388-1390, 1991; Clem and Miller, Mol. Cell. Biol. 14:5212-5222, 1994). Inappropriate (or premature) apoptosis may contribute to neurodegenerative disorders (Roy et al., Cell 80:167-178, 1995; Raff et al., Science 262:695-700, 1996) or acquired immunodeficiency disease (Banda, N., J. Exp. Med. 176:1099-1106, 1992). Premature apoptosis is also recognized as a contributing cause of myocyte loss in ischemia/repurfusion injury, myocardial infarction (MacLellan and Schneider, Circ. Res. 81:137-144, 1997), and congestive heart failure (Feuerstein, G., Trends Cardiovas. Med. 7:249-255, 1997).
The presence of a novel class of apoptosis inhibitors, known as inhibitor of apoptosis proteins (IAPs) has been reported in the literature (Liston et al., Apoptosis 2:423-441, 1997). The first IAP was discovered in baculovirus (Crook et al., J. Vir. 67:2166-2174, 1993) and IAPs have now been reported in Drosophila, chick, mouse and human (Hay et al., Cell 83:1253-1262, 1995; Liston et al., supra). Five human IAPs have been identified: HIAP1, HIAP2, XIAP (X-chromosome linked IAP), NIAP (neuronal IAP) and survivin (Ambrosini et al., Nat. Med. 3:917-921, 1997; Duckett et al., Embo J. 15:2685-2694, 1996).
IAPs are a highly evolutionarily conserved family of proteins, containing a number of common structural features (domains). Among these are an N-terminal domain containing one or more repeats of a domain referred to as the BIR (baculovirus IAP repeat) domain (Liston et al., supra), and a C-terminal RING zinc finger domain. These domains are present to varying degrees within the known members of the IAP family; HIAP1 and HIAP2 contain three BIR domains and a C-terminal RING domain, while survivin contains only a single BIR domain and no RING domain.
While the physiological role of IAPs is not exactly clear, some members of the IAP family appear to play a regulatory role in apoptosis. Recombinant IAPs were found to suppress apoptosis induced by a variety of stimuli in different cell types. Drosophila IAPs (DIAP1 and DIAP2) were found to interact with a Decapentaplegic (Dpp) type I receptor, suggesting that these DIAPs may act as negative regulators of the Dpp signaling pathway, which normally leads to cell apoptosis. XIAP, HIAP1 and HIAP2 can directly inhibit specific caspases (cysteine containing aspartate specific proteases), enzymes which are involved in the pathways which control apoptosis, and thereby suppress apoptosis (Thornberry, N., Br. Med. Bull. 53:478-490, 1997). However, NIAP was found not to inhibit caspases, suggesting that different IAPs may have different mechanisms of action.
By helping in the regulation of programmed cell death, IAPs play an important role in the maintenance of the appropriate life cycle of the various cells of an organism. It is likely that variance from normal levels (either overabundance or deficiency) of IAPs within the cellular environment may lead to conditions in vivo which are related to various disease states.
IAPs may play a role in tumor formation. Up-regulated chicken IAP and concommitant suppression of apoptosis were found in chicken cells transformed by the oncoprotein v-rel, a member of the Rel/NFkappaB family (You et al., Mol. Cell. Biol. 17:7328-7341, 1997). Similarly, baculovirus protein p35 (a baculovirus IAP) is capable of promoting the transformation of mouse embryo fibroblasts in the presence of the insulin-like growth factor I receptor (Resnicoff et al., J. Biol. Chem. 273:10376-10380, 1998).
Survivin is undetectable in terminally differentiated adult tissues but expressed in all common human cancers, further suggesting that apoptosis inhibition may be a general feature of neoplasia (Ambrosini et al., supra).
Deletion mutations in human NIAP have been linked to inappropriate depletion of motor neurons associated with spinal muscular atrophy, an autosomal neurodegenerative disorder (Xu et al., J. Comp. Neurol. 382:247-259, 1997). In a rat ischemia model, in vivo overexpression of NIAP reduced ischemic damage in the rat hippocampus (Roy et al., supra), indicating that the presence of increased levels of IAPs could prevent the unwanted cell death characteristic of ischemia and that elevating the neuronal levels of this IAP may be useful in treating stroke.
Finally, Stellar and his colleagues were able to block retinal cell death and show significant retention of visual function in Drosophila which exhibited retinitus pigmentosa, a cause of blindness in humans, by eye-specific expression of the antiapoptotic protein p35 (Davidson and Stellar, Nature 391:587-591, 1998).
These data suggest that antiapoptotic proteins, such as IAPs, are good candidates for use in the therapeutic intervention of diseases caused by altered apoptosis.
There is a need, therefore, for identification and characterization of proteins that influence apoptosis. In particular, there is a need to isolate and characterize additional IAPs, akin to known IAPs, which may be employed, therefore, for ameliorating or correcting dysfunctions or disease associated with inappropriate apoptosis; in cancer and chronic viral infections, where IAPs may be overproduced, as well as in neurodegenerative disorders, chronic heart failure and dysfunctional immune response, where a deficiency in IAPs may exist.
The present invention provides a polynucleotide sequence which uniquely encodes a novel human inhibitor-of-apoptosis protein. Designated HIAP3, the polypeptide is characterized by structural features common to the inhibitor-of-apoptosis protein family, such as BIR and RING domains. The polynucleotide sequence, designated in lower case, hiap3, and described in FIG. 1 (SEQ ID NO:1) encodes the amino acid sequence, which is designated HIAP3, and is shown in FIG. 2 (SEQ ID NO:2).
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as a novel HIAP3 by homology between the amino acid sequence set out in FIG. 2 (SEQ ID NO: 2) and known amino acid sequences of other IAP proteins.
It is a further object of the invention, moreover, to provide polynucleotides that encode HIAP3, particularly polynucleotides that encode the polypeptide herein designated HIAP3.
In accordance with this aspect of the invention there are provided isolated polynucleotides encoding HIAP3, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of hiap3 polynucleotides.
It also is an object of the invention to provide HIAP3 polypeptides that may be employed to treat neoplasia, neurodegenerative disorders, immune disorders, chronic viral infections, or chronic heart failure.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as HIAP3 as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
Among the particularly preferred embodiments of this aspect of the invention are variants of HIAP3 encoded by naturally occurring allelic variants of the hiap3 polynucleotide.
It is another object of the invention to provide a method of producing the aforementioned polypeptides, polypeptide fragments variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods of producing the aforementioned HIAP3 polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived HIAP3-encoding polynucleotide under conditions for expression of human HIAP3 in the host and then recovering the expressed polypeptide.
In accordance with another object of the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for, among other things: assessing HIAP3 expression in cells by determining HIAP3 polypeptides or HIAP3-encoding mRNA; assaying genetic variation and aberrations, such as defects, in hiap3 genes; and administering an HIAP3 polypeptide or polynucleotide encoding HIAP3 to an organism to alter the level of HIAP3 activity.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize to hiap3 sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against HIAP3 polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for HIAP3, and may be employed diagnostically to detect increased HIAP3 expression, which may be associated with conditions in which inhibition of apoptosis is too strong, such as cancer.
In a further aspect of the invention there are provided compositions comprising an hiap3 polynucleotide or an HIAP3 polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise an hiap3 polynucleotide for expression of an HIAP3 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a disease state which is alleviated by increasing the level of HIAP3 activity.
In a further aspect of the invention there are provided ribozymes and polynucleotides complementary to hiap3 polynucleotides (i.e. antisense polynucleotides) for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. Particularly preferred in this regard is administration to a human patient for treatment of a disease state which is alleviated by decreasing the level of HIAP3 activity.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following. description and from reading the other parts of the present disclosure.